Transformed embryogenic microspores for the generation of fertile homozygous plants

ABSTRACT

The invention relates to transformed, embryogenic microspores and progeny thereof characterized by being transformed by  Agrobacterium tumefaciens , capable of leading to non-chimeric transformed haploid or doubled haploid embryos that develop into fertile homozygous plants within one generation and containing stably integrated into their genome a foreign DNA, said DNA being characterized in that it comprises at least one gene of interest and at least base pairs within the right border sequence of Agrobacterium T-DNA. The invention furthermore relates to a method for the incorporation of foreign DNA into chromosomes of microspores comprising the following steps: a) infecting of embryogenic microspores with Agrobacteria, which contain plasmid carrying a gene of interest under regulatory control of initiation and termination regions bordered by at least one T-DNA border, b) Washing out and killing the Agrobacteria after co-cultivation.

1. INTRODUCTION

Transgenic crop plants can be produced using different transformationtechniques. The method of choice for species and varieties, where plantregeneration from tissue culture is efficient, is Agrobacterium(Potrykus 1993). The Agrobacterium tumefaciens transformation system issimple and inexpensive (DeBlock 1993). It is the most effective methodof transferring foreign DNA into a host plant (Thierfelder et al. 1993).Plants are obtained with a limited number of gene insertions, andusually insertion of DNA occurs between two defined border sequencescompletely and exclusively (DeBlock 1993). It is therefore notsurprising that in terms of use and apparent progression to fieldtrials, the majority of gene transfer experiments have involved theAgrobacterium system (White 1993).

Microspores of higher plants have the potential to develop, underappropriate conditions directly into haploid or doubled haploid plants.Microspores of higher plants develop in vivo into pollen grains(gametophytic pathway). Microspore culture can induce an alternative(sporophytic) pathway which leads to the formation of haploid anddoubled haploid embryos. These embryos develop directly into normalfertile plants. Misunderstandingly these embryos are sometimes referredto as pollen embryos.

The time saving for the breeder working with microspore transformantscompared to transformants from heterozygous plant material is estimatedto be three years for the development of each transgenic variety. Thisis due to the immediate fixation of the genes in the homozygous doubledhaploid lines. This allows a more efficient selection of agronomic andquality traits and the immediate testing of combining ability for thedevelopment of hybrid cultivars.

Therefore, since embryogenic microspores develop into homozygous fertileplants and are single haploid cells, they are theoretically the mostsuitable recipients for gene transfer (Huang and Keller 1989).

Several laboratories (Potrykus 1991, Heberle-Bors 1995) haveunsuccessfully attempted transforming microspores with Agrobacterium andSangwan et al. (1993) have concluded that “it is difficult if notimpossible to obtain transgenic plants by infection of pollen orproembryos with Agrobacterium.” Interestingly Heberle-Bors (1995)summarizes his attempts to transform microspores stating that A.tumefaciens was not suitable to transfer DNA into microspores, contraryto his 1989 patent. The thick cell wall of microspores has beenperceived to be inaccessible to Agrobacterium (Stöger et al. 1995,Jones-Villeneuve et al. 1995).

More frequently, multicellular haploid tissues such asmicrospore-derived embryos; (Neuhaus et al. 1987, Swanson and Erickson1989, Huang 1992) or haploid stem segments (Arnoldo 1992) have beenused.

A common misconception is the difference between microspores andmicrospore-derived embryos where doubled-haploid plants can be obtainedfrom both. Microspores are single cells and can be stimulated to produceembryos. The transformation of microspores leads to non-chimeric embryosand then plants. On the other hand microspore-derived embryos aremulti-celled tissues. The transformation of microspore-derived embryosegments may lead to chimeric sectors from which chimeric shoots orsecondary embryos can be selected. In addition somaclonal variation isnot uncommon as there is usually an intermediate callus phase associatedwith regeneration from microspore-derived embryo segments.

The closest report of the prior art is the publication of Pechan (1989)on microspore co-cultivation with Agrobacterium. He claimed theproduction of kanamycin and hygromycin resistant plants but did notprove DNA integration or the sexually transmissibility of the transgeneand furthermore, the described method has not been reproducible byothers (Huang 1992).

Several reports describe the use of transformation techniques onmicrospores. Huang (1992) reported the use of microinjection, particlebombardment and electroporation on microspores and observed sometransient gene expression, but no stable transformants. The review alsodescribed the use of Agrobacterium on microspores and in over 50experiments conducted, only one plant was regenerated where Agrobacteriawere present at the proembryo stage and the event could not bereproduced. Jardinaud et al. (1993) reported only transient geneexpression in experiments using electroporation on B. napus microsporesand using biolistics on maize microspores (Jardinaud 1995).Microinjection has also been attempted in B. napus microspores but thiswas not successful (Jones-Villeneuve et al. 1995).

Particle bombardment is the only method used on embryogenic microsporesthat has led to the production of stable transformed plants (Jähne etal. 1994, Stöger et al. 1995). However in comparing differenttransformation methods, Christou (1995) concluded that with biolistics,the underlying mechanism of the gene transfer process is not known andthe impossibility to control DNA content and integration patterns is amajor drawback. Transformed plants normally have multi-copy (Jähne etal. 1994) and random fragmented DNA insertions, that may or may notcontain the complete gene of interest.

For breeding purposes, single copy gene insertions are desirable as theycan be selected easily and followed through generations with ease andgene expression is good. In addition the plant does not need totranscribe more than the necessary enzymes and phenomena such asco-suppression, i.e. the down regulation of a gene by additional copiesof this gene having substantial homology (WO 90/12084), as wastes ofvaluable plant energy is not a consideration.

The optimal transformation vector to allow single copy gene insertionswith defined border sequences is to make use of the naturally occuringtransformation process of Agrobacterium tumefaciens.

We surprisingly found that microspore transformation via A. tumefaciensis practicable and leads to fertile homozygous plants with predominantlysingle copy inserts which have been confirmed via Southern blots in B.rapa and B. napus genotypes. Thus fertile homozygous plants with singlecopy inserts can be produced in only one gene ration using embryogenicmicrospores and A. tumefaciens.

2. DESCRIPTION OF THE INVENTION

The present invention is directed to transformed embryogenicmicrospores. The invention additionally provides an improved process forthe Agrobacterium mediated transformation.

The invention is directed to transformed, embryogenic microspores andprogeny thereof characterized by

a) being transformed by Agrobacterium tumefaciens,

b) capable of leading to non-chimeric transformed haploid or doubledhaploid embryos that develop into fertile homozygous plants within onegeneration and

c) containing stably integrated into their genome a foreign DNA, saidDNA being characterized in that it comprises at least one gene ofinterest and at least base pairs within the right border sequence ofAgrobacterium T-DNA.

The invention is furthermore directed to microspores containingintegrated foreign DNA wherein the gene of interest is bordered by atleast base pairs within the right border and parts or all of the leftborder sequence leading to the specified insertion of the gene(s) ofinterest. Especially preferred are microspores containing at least oneinsert of said foreign DNA.

The invention also encompasses transformed, embryogenic microsporescapable of being produced according to a process comprising thefollowing steps:

a) infection of embryogenic microspores with Agrobacteria

b) washing out and killing the Agrobacteria after co-cultivation duringtransformation.

Preferrably, the Agrobacteria are removed by washing with mucolyticenzymes after co-cultivation and the process comprises the further stepof adding cellolytic enzymes during transformation.

The invention is also directed to a method for the incorporation offoreign DNA into chromosomes of microspores comprising the followingsteps:

a) infecting of embryogenic microspores with Agrobacteria, which containplasmids carrying a gene of interest under regulatory control ofinitiation and termination regions bordered by at least one T-DNAborder,

b) washing out and killing the Agrobacteria after co-cultivation.

Especially preferred is a method wherein the Agrobacteria are removed bywashing with mucolytic enzymes after co-cultivation, and in which thegene of interest is bordered by at least one T-DNA border as well as amethod leading to the specific insertion of the gene of interest.

The invention further comprises transgenic microspore derived plantscomprising a foreign genetic construct inserted into the microspores bythe method described above.

According to the invention the transformed embryogenic microsporesdevelop into transgenic homozygous fertile plants within one generation.In a preferred embodiment of the invention the microspores carry onlyfew copy inserts, especially preferred one to three inserts, mostfavourably only a single copy insert.

One of the major advantages of the process according to instantinvention is the transfer of a defined sequence starting within theright border sequence of the T-DNA.

More particularly the present invention covers transformed embryogenicmicrospores which develop into plants and are characterized by:

a. Being transformed by A. tumefaciens

b. Expressing mainly single copy inserts

c. Leading to non-chimeric transformed haploid or doubled haploid(homozygous) embryos that develop into fertile plants within onegeneration.

The transformed, embryogenic microspores are obtainable by a processcomprising the following steps:

a infection of embryogenic microspores with Agrobacteria

b addition of cellolytic enzymes during Agrobacterium co-cultivation

c wash with mucolytic enzymes after co-cultivation to kill theAgrobacteria to increase the number of surviving transformants.

The term cellolytic enzyme used hereafter refers to any substance whichdissolves cell wall components of the explant inducing a wound responseor detergent effect which faciliates gene transfer, i.e. cellulase,hemicellulase, pectinase.

The term mucolytic enzyme used hereafter refers to any substance whichdissolves prokaryotic cell wall components (mureines, peptidoglycanes),i.e. lysozyme.

The cellolytic enzymes applied during cocultivation are selected onbasis of their capacity of a.) to increase the efficiency oftransformation (detergent effect) and b.) to increase the yield ofrecoverable transformed plantlets. Besides improving transformationefficiency they help to overcome the detrimental effect of longerco-cultivation with Agrobacteria (aggregation and overgrowth ofexplants) by dissolving selectively the fibrils produced byAgrobacterium during the coculture.

In a preferred embodiment the invention covers transgenic embryogenicmicrospores produced by the process involving cellulase and lysozyme toenhance the transformation efficiency.

The solutions containing the lytic enzymes contain preferably from about0.0004 to about 0.1%, most preferably from about 0.004 to about 0.01% ofthe cellulose and preferably from about 0.1 to about 10%, especiallyprefered from about 0.5 to about 5% and most preferably from about 0.75to 1.5% lysozyme.

The concentrations to be used depend on the length of incubation. Theused cellulase concentration apply to an incubation period of 3 days.Accordingly, higher concentrations may be used if the incubation periodis shorter.

The active enzymes used according the present invention are normallyapplied in form of compositions together with one or more acceptablesupplements, and car be applied to the embryogenic microspores to betreated, simultaneously or in succession, with further compounds.

These compounds can be antimicrotubule active compounds like colchicine(Möller et al. 1994) or trifluralin (Zhao and Simmonds 1995) that leaddirectly to an in vitro duplication of the haploid microspore genome,which may additionally affect transformation efficiency by synchronizingthe cell cycle of the microspores.

They can also be buffer substances or mixtures of several of thesepreparations, if desired together with further medium supplementscustomaily employed in the art of transformation.

Suitable media can be semisolid or liquid and correspond to thesubstances ordinarily employed in transformation technology, e. g. B5medium (GAMBORG et al. 1968).

The number of applications and the rate of application depend on theAgrobacterium strain and its culture, the time and temperature duringcocultivation and the microspore material, i.e. the species orcultivars.

Another object of this invention is to provide a new process fortransforming embryogenic microspores which comprises at least oneadditional washing step with mucolytic enzymes after co-cultivation withAgrobacterium. The washing procedure may be performed preferably asdescribed in materials and methods. Said washing is repeated preferablyone to two times.

The solution which may be used to wash the embryogenic microspores maybe especially in the form of Tris HCL (pH 8.0) as described in Sambrook,Fritsch & Maniatis (1989).

A preferred method of introducing the nucleic acid segments intoembryogenic microspoes is to infect microspores with A. tumefacienscarrying an inserted DNA construct (see especially U.S. Pat. No.5.188.958 and EP 0 270 615 B1). The nucleic acid segments or constructscan be introduced into appropriate plant cells, for example, by means ofthe Ti plasmid of A. tumefaciens. The T-DNA is transmitted toembryogenic microspores upon infection by A. tumefaciens, and is stablyintegrated into the plant genome. Under appropriate conditions known inthe art, the transformed embryogenic microspores develop further intoplants.

The Agrobacterium strains customarily employed in the art oftransformation are described, for example, in White (1993).

Ti plasmids contain two regions essential for the production oftransformed cells. One of these, named transfer DNA (T DNA), inducestumor formation. The other, termed virulent region, is essential for theintroduction of the T DNA into plants. The transfer DNA region, whichtransfers to the plant genome, can be increased in size by the insertionof the foreign nucleic acid sequence without its transferring abilitybeing affected. By removing the tumor causing genes so that they nolonger interfere the modified Ti plasmid (“disarmed Ti vector”) can thenbe used as a vector for the transfer of the gene constructs of theinvention into embryogenic microspores. In the binary system, to haveinfection, two plasmids are needed: a T-DNA containing plasmid and a virplasmid. Any one of a number of T-DNA containing plasmids can be used,the only requirement is that one be able to select independently foreach of the two plasmids (see especially EP 116718 B1 and EP 120 516B1).

The embryogenic microspores with the integrated desired DNA sequence canbe selected by an appropriate phenotypic marker. These phenotypicmarkers include, but are not limited to, antibiotic resistance,herbicide resistance or visual observation. Other phenotypic markers areknown in the art and may be used in this invention.

The DNA constructs used in instant invention consist of a transcriptioninitiation region and, under the control of the transcription initiationregion, a DNA sequence to be transcribed. The DNA sequence may comprisea natural open reading frame including transcribed 5′ and 3′ flankingsequences. Alternatively, it may comprise an anti-sense sequence thatencodes the complement of an RNA molecule or portion thereof (asdesribed in EP 140 308 B1 and EP 223 399 B1).

The initiation regions may be used in a variety of contexts and incombination with a variety of sequences. The RNA coded sequences of agene may be those of a natural gene, including the open reading framefor protein coding and frequently the 5′ and 3′ untranslated sequences.The RNA translational initiation sequences are included in theconstructs, either from the promoter domain or from the attached codingsequences.

Attached to the above sequences are appropriate transcriptiontermination and polyadenylation sequences.

Examples of the above sequences or genes to be expressed from theconstructs of the subject invention include:

antisense or sense genes (for gene suppression);

genes for nutritionally important proteins: growth promoting factors,yield enhancing factors, e.g. an asparagine synthetase gene or aninvertase gene;

genes for proteins giving protection to the plant under certainenvironmental conditions, e.g. proteins giving resistance to metal orother toxicity;

genes for stress related proteins giving tolerance to extremes oftemperature, freezing, etc.

genes for proteins of specific commercial value;

genes causing increased level of proteins, e. g., enzymes of metabolicpathways,

genes causing increased levels of products of structural value to aplant host, e.g., herbicide resistance, fungus resistance, e.g.chitinase genes, glucanase genes, proteins synthesis inhibitor genes,ribosome inhibitory protein genes, viral resistance, e.g. ribozymes,virus coat protein genes.

The subject constructs will be prepared employing cloning vectors, wherethe sequences may be naturally occuring, mutated sequences, syntheticsequences, or combinations thereof. The cloning vectors are well knownand comprise prokaryotic replication systems, markers for selection oftransformed host cells, and restriction sites for insertion orsubstitution of sequences. For transcription and optimal expression, theDNA may be transformed into embryogenic microspores for integration intothe genome, where the subject construct is joined to a marker forselection or is co-transformed with DNA encoding a marker for selection.

The selection of transformed embryogenic microspores is enabled by theuse of a selectable marker gene which is also transferred. Theexpression of the marker gene confers a phenotypic trait that enablesthe selection. Examples for such genes are those coding for antibioticsor herbicide resistance, e.g. genes causing resistance against glutaminesynthetase inhibitors, e.g. bialaphos or phosphinothricin resistanceconferred by genes isolated from Streptomyces hygroscopicus oriridochromogenes (BAR/PAT). Other examples are the neomycinphosphotransferase or the glucuronidase gene.

The process claimed in instant invention provides a comparable number ofeffectively transformed embryogenic microspores. The efficiency oftransformation was about two up to 15 percent. As efficiency we definehere the number of transformed embryos (plants) related to the number ofall embryos (plants) plated on the selection medium.

The transformation method using Agrobacterium is relatively simple,highly reproducible and has a high possibility of being extrapolated toother embryogenic microspore culture systems. In all species wheremicrospores can developed into embryos (androgenesis), this method canbe expected to generate transgenic haploid or doubled haploid plants ina one step procedure.

The class of transgenic embryogenic microspores which are covered bythis invention is generally as broad as the class of higher plantsamenable to androgenesis (development from microspores to embryos thatdevelop into plants) and techniques developed to stimulate androgenesisincluding both monocotyledonous and dicotyledonous plants. It is knownthat theoretically all plants can be regenerated from culturedembryogenic microspores, including but not limited to all major cerealcrop species, sugarcane, sugar beet, cotton, fruit and other trees,legumes and vegetables.

Accordingly, the use of embryogenic microspores will help to producetransformed plants in genotypes of cereal and legume species which arenow considered recalcitrant. Although it has been shown that for examplewheat, rice, maize can be transformed with Agrobacterium, broad successhas been impeded greatly by the difficulty to direct the gene transfertowards plant cells that are amenable to regeneration (van Rordragen &Dons 1992). With increased knowledge about the biology of the infectionprocess of Agrobacterium (Smith & Hood 1995) and the rapid developmentin cereal microspore culture, the use of embryogenic microspores will beespecially suitable for the transformation of monocotyledons (Jähne andLörz 1995). In the meantime it has been shown that even yeast can betransformed with A. tumefaciens (Piers et al. 1996).

Examples of families that are of special interest are Poaceae, but alsoSolanaceae and Brassicaceae.

Some suitable species include, for example, species from the generaFragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciohorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia,Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis. Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Examples of species of commercial interest that can be protectedinclude:

tobacco, Nicotiana tabacum L.

tomato, Lycopersicon esculentum Mill,

potato, Solanum tuberosum L.,

Canola/Rapeseed,

Brassica napus L.,

cabbage, broccoli, kale etc.,

Brassica oleracea L.,

mustards Brassica juncea L.,

Brassica nigra L.,

Sinapis alba L. (Brassicaceae),

petunia, Petunia hybrida (Solanaceae)

sugar beet, Beta vulgaris, (Chenopodiaceae),

cucumber, Curcurbita sp. (Curcurbitaceae),

cotton, Gossypium sp., (Malvaceae),

sunflower, Helianthus annuus,

lettuce Lactuca sativa, (Asteraceae=Compositae),

pea, Pisum sativum,

soybean, Glycine max and alfalfa, Medicago sp. (Fabaceae=Leguminoseae),

asparagus, Asparagus officinalis;

gladiolus, Gladiolus sp., (Lilaceae);

corn, Zea mays;

rice, Oryza sativa (Poaceae);

wheat, Triticum aestivum (Poaceae); and

barley, Hordeum vulgare (Poaceae).

In an preferred embodiment the invention covers transformed embryogenicmicrospores in the Brassica species for example B. napus, B. rapa, B.juncea, B. oleracea, B. carinata and others.

The invention additionally relates to Brassica plants which have beenregenerated out of embryogenic microspores which have been transformedaccording to instant invention.

The present invention is directed to the use of embryogenic microsporesfor a new haploid transformation system. Microspores refers to freshlyisolated microspores up to 72 hours after isolation which comprisemainly of single cells as the first cell division is usually visiblethree days after culture. The microspore system offers advantages overother explant methods in that regeneration leads to the production ofhaploid plants or homozygous diploid plants. This offers simplicity forgenetic studies and one-step fixation of genes for breeding purposes.

The use of embryogenic microspores for transformation can be carried outas described in the materials and methods and in the examples.

In general, preparation of plasmid DNA, restriction enzyme digestion,agarose gel electrophoresis of DNA, Southern blots, DNA ligation andbacterial transformation were carried out using standard methods.(Sambrook I. et al. (1989), referred to herein as “Maniatis” and herebyincorporated by reference.)

3. MATERIALS AND METHODS

The following disclosure describes the use of embryogenic microsporesand the tranformation process. The disclosure will then be completedwith the description of the conditions under which embryogenicmicrospores can be transformed, also merely by way of examples fornon-limitative illustration purposes.

A. tumefaciens cells (MP90RK with pHoe6Ac, see FIG. 1) (diluted 10⁻⁴ to10⁻⁷ times from a 0.9 O.D. spectrophotometric standard) were added tofreshly isolated microspores or microspores cultured up to 72 h. Adilution of 10⁻⁵ corresponds to a concentration of approximately 10,000Agrobacteria per ml. The cultures were supplemented with cellolyticenzymes and incubated for 72 hours following the temperature regimedescribed by Baillie et al. (1992).

For one experiment usually 5 petridishes have been filled withmicrospores harvested from about three plants. One petri dish containsapproximately the microspores from 10 flower buds.

Subsequently (after three days of cocultivation), the cultures werewashed gently by discarding the supernatant and adding fresh NLN mediumcontaining 10% sucrose sucrose with 500 mg/l carbenicillin (or 300 mg/lTimentin). As the microspores adhere tightly to the bottom of the petriplate at this stage of infection, there is very little loss ofmicrospores.

The microspores adhering to the bottom of the petri plates are thengently scraped and loosened from the bottom of the plate using softrubber policemen (Fisher Scientific). Then the fresh medium andmicrospores were transferred into 50 ml Falcon tubes for centrifugation.

Centrifugation was carried out two times for three minutes (at 200 g).Microspores were suspended and centrifuged at least once in lysozyme and10 mM Tris HCL. buffer pH 8.0. Finally fresh NLN medium plus 10% sucroseand 500 mg/l carbenicillin (or 300 mg/l Timentin) was added and thecultures were incubated according to Baillie et al. 1992.

The cultures are kept at 24° C. until they are four weeks old as thedeveloping embryos are usually a week slower than non-infected controlembryos.

Selection for true transformants can be started as soon as embryosbecome visible, the selection is preferably carried out after greeningof the embryos has started.

The timeframe from microspore isolation until transformed plantlet inthe greenhouse takes approximately three months.

The following examples are intended only to further illustrate theinvention and are not intended to limit the scope of the invention whichis defined by the claims.

4. EXPERIMENTAL DATA 4.1. Optimization of Co-cultivation Conditions forImproved Embryo Recovery and Transformation Efficiency

Material: Fresh buds of Brassica rapa, CV2

DNA construct: glufosinate (Phosphinothricin, L-PPT) resistance geneunder control of 35S promotor and 35S terminator (Plasmid pRD 320 havingthe same sequence as pRD 420 (Datla et al. 1992) but with the PAT gene).

Preculture: Incubator (10/5° C., 16 h photoperiod)

A Treatment (Control) B C D E day 0  Initiaton Infection InfectionInfection Infection of micro- with without with without spore cellulasecellulase cellulase cellulase culture day 2  Transfer Transfer TransferTransfer Transfer day 3  centrifuge centrifuge hand wash centrifuge handwash wash wash wash day 23 490 12 16 18 24 embryos/ embryos/ embryos/embryos/ embryos/ plate plate plate plate plate day 30 greening greeninggreening greening greening day 39 plating plating plating platingplating day 54 rooting rooting rooting rooting rooting assay assay assayassay assay day 59 0 10 4 11 6 A: Control (uninfected) B: + cellulase +Agrobacterium (10⁻⁶) C: + cellulase + Agrobacterium (10⁻⁶) D: +cellulase + Agrobacterium (10⁻⁷) E: − cellulase + Agrobacterium (10⁻⁷)day 0: Initiation of microspore culture after Baillie (1992) but withoutamino acids. Infection with A. tumefaciens and addition of cellulasewhere applicable. Incubation for two days at 32° C. day 2: Transfer ofplates to 24° C. incubator. day 3: Media change to NLN10. Centrifugewash was performed 2 times at 200 g. day 23: Embryo count. day 30:Greening up of embryos under continous light at 22° C. on shaker at 75rpm. day 39: Plating on OB5 solid medium. day 54: Start L-PPT rootingassay (Selection on 20 mg/l L-PPT). day 59: Transfer of putativetransformants to OB5 solid medium.

The data for the recovered embryos and putative transformants aresummarized in the attached graphics. The results show that the additionof cellulase does not only dissolve the fibrils of the Agrobacteria toenable centrifuge wash, but also increased transformation efficiency,obviously by dissolving cellulose of the plant material. Cellulase onthe other hand reduced the number of embryos recovered slightly withineach level of the tested Agrobacterium concentration.

4.2. Comparison of the Effect of Different Washing Procedures on EmbryoYield After Infection with A. tumefaciens

Material: Buds of Brassica rapa, CV2

DNA construct: pHoe6Ac (see FIG. 1, AC stand for Acetyltransferase, i.e.PAT)

Preculture: Incubator (10/5° C., 16 h photoperiod), flower buds storedat 4° C. for two days prior to micorspore culture.

A (Control) B C D E day 0 Initiation Initiation Initiation InitiationInitiation + day 3 transfer Infection Infection Infection Infection andand and and transfer transfer transfer transfer day 5 — — — +cellulase + cellulase day 6 Centrifuge Centrifuge Hand CentrifugeCentrifuge wash wash wash wash wash + media media media media lysozymechange change change change media and (lots of (fibrils change lysozymeconnected dissolved) (fibrils treatment fibrils) dissolved) day 22 484390 no 27 58 embryos/ embryos/ embryos embryos/ embryos/ plate plateplate plate A: uninfected control, B: uninfected control and theapplication of lysozyme during media change, C: hand wash at day 6, D:Addition of cellulase at day 5 and centrifuge wash at day 6, E: as D butwith additional lysozyme treatment at day 6. day 0: Initiation ofmicrospore culture after Baillie (1992) but without amino acids.Incubation for two days at 32° C. day 3: Infection with Agrobacteriumand transfer of plates to 24° C. incubator. day 5: Addition ofcellulase. day 6: Wash: Centrifuge wash where applicable at 200 g.Lysozyme treatment where applicable. Media change to NLN10 in alltreatments. day 22: Embryo count: Lysozyme treatment doubled the numberof recovered embryos. Mean number of embryos of E differs significantlyfrom C (tested with 8 replicates per treatment).

4.3. Generation of Transformants Carrying Disease Reistance Genes

Material: Brassica napus, Topaz 4079

DNA construct: pGJ57 (Glucanase, Chitinase, PAT each with 35S terminatorand 35 S promoter, see FIG. 2)

Preculture: Incubator (10/5° C., 16 h photoperiod), flower buds, storedat 4° C. for two days prior to initiation of microspore culture.

A (Control) B day 0 Initiation of Initiation of microspore culture^(x1)microspore culture day 1 — Infection with Agrobacterium day 3 TransferTransfer day 4 Centrifuge wash Centrifuge wash + lysozyme treatment day22 422 embryos/plate 70 embryos/plate day 60 50 healthy plants 50healthy plants day 66 0 2^(x2) ^(x1)Each treatment comprises fiveplates. Per plate approximately ten flower buds have to be harvested^(x2)Fifty healthy plants were transferred to selection medium. Out offifty two independent transformants were selected. This represents atransformation frequency of 4%. The two transformants were confirmed viaSouthern analysis. A: uninfected control, B: As A but infected with A.tumefaciens (10⁻⁶) day 0: Initiation of microspore culture. Incubationfor two days at 32° C. day 1: Infection with A. tumefaciens whereapplicable and addition of colchicine day 3: Transfer of plates to 24°C. incubator day 4: Centrifuge wash was performed 2 times at 200 g.Lysozyme treatment. Media change to NLN10. day 22: Embryo count. day 60:Start L-PPT Rooting Assay (20 mg/l L-PPT). day 66: Transfer of putativetransformants to OB5 solid medium.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modification and variations may be madetherin without departing from the spirit and scope of the invention.

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6. FIG. 2—LEGEND

Molecular Features Type Start End Compl Name Description GENE  790  1758Sm/Sp strep/spec adenyltransferase GENE  5611  5634 LB left border fromoctopine Ti ACH5 GENE  6123  5917 (C) T-35S CaMV 35S terminator GENE 6693  6142 (C) PAT synthetic PAT gene GENE  7251  6722 (C) P-35S CaMV35S promoter GENE  7519  7324 (C) T-35S 35S terminator GENE  8506  7706(C) chitinase chitinase GENE  9000  8582 (C) P-35S 35S promoter GENE 9216  9021 (C) T-35S 35S terminator GENE 10483  9467 (C) glucanaseglucanase GENE 10949 10531 (C) p-35S 35S promoter GENE 11173 11196 RBright border from pTiT37

What is claimed is:
 1. A method for producing a stably transformed Brassica embryogenic microspore, capable of leading to a non-chimeric transformed haploid or doubled haploid embryo which develops into a fertile homozygous Brassica plant within one generation, said process comprising the following steps: a. infecting an embryogenic microspore with Agrobacteria, which contain a plasmid carrying a gene of interest under regulatory control of initiation and termination signals bordered by at least one T-DNA border, and b. washing out and killing the Agrobacteria after co-cultivation using mucolytic enzymes, thereby producing a stably transformed Brassica embryogenic microspore.
 2. A method for producing a non-chimeric Brassica plant, containing a foreign DNA stably incorporated into its genome, said method comprising: a. co-cultivating a Brassica embryogenic microspore with Agrobacteria which contains a plasmid carrying a gene of interest under regulatory control of initiation and termination signals; b. washing out and killing the Agrobacteria after co-cultivation using mucolytic enzymes; and c. regenerating a non-chimeric haploid or doubled haploid Brassica embryo from said microspore, wherein the embryo contains said gene of interest stably integrated into its genome, thereby producing a non-chimeric Brassica plant.
 3. The method according to claim 1, further comprising the step of adding cellolytic enzymes during Agrobacterium co-cultivation.
 4. The method according to claim 3, wherein said cellolytic enzyme is a cellulase.
 5. The method according to claim 1, wherein the mucolytic enzyme is a lysozyme.
 6. The method according to claim 2, further comprising the step of adding cellolytic enzymes during Agrobacterium co-cultivation.
 7. The method according to claim 6, wherein said cellolytic enzyme is a cellulase.
 8. The method according to claim 2, wherein the mucolytic enzyme is a lysozyme. 